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Link to original content: http://pubmed.ncbi.nlm.nih.gov/38706988/
Bortezomib promotes the TRAIL-mediated killing of resistant rhabdomyosarcoma by ErbB2/Her2-targeted CAR-NK-92 cells via DR5 upregulation - PubMed Skip to main page content
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. 2024 Apr 11;32(2):200802.
doi: 10.1016/j.omton.2024.200802. eCollection 2024 Jun 20.

Bortezomib promotes the TRAIL-mediated killing of resistant rhabdomyosarcoma by ErbB2/Her2-targeted CAR-NK-92 cells via DR5 upregulation

Catrin Heim  1   2 , Leonie Hartig  1   2 , Nadine Weinelt  3 , Laura M Moser  1   4   5   6   2 , Emilia Salzmann-Manrique  1   2 , Michael Merker  1   6   2 , Winfried S Wels  4   5   7 , Torsten Tonn  8 , Peter Bader  1   6   2 , Jan-Henning Klusmann  4   5   6   2 , Sjoerd J L van Wijk  3   4   6 , Eva Rettinger  1   4   5   6
Affiliations

Bortezomib promotes the TRAIL-mediated killing of resistant rhabdomyosarcoma by ErbB2/Her2-targeted CAR-NK-92 cells via DR5 upregulation

Catrin Heim et al. Mol Ther Oncol. .

Abstract

Treatment resistance and immune escape are hallmarks of metastatic rhabdomyosarcoma (RMS), underscoring the urgent medical need for therapeutic agents against this disease entity as a key challenge in pediatric oncology. Chimeric antigen receptor (CAR)-based immunotherapies, such as the ErbB2 (Her2)-CAR-engineered natural killer (NK) cell line NK-92/5.28.z, provide antitumor cytotoxicity primarily through CAR-mediated cytotoxic granule release and thereafter-even in cases with low surface antigen expression or tumor escape-by triggering intrinsic NK cell-mediated apoptosis induction via additional ligand/receptors. In this study, we showed that bortezomib increased susceptibility toward apoptosis in clinically relevant RMS cell lines RH30 and RH41, and patient-derived RMS tumor organoid RMS335, by upregulation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor DR5 in these metastatic, relapsed/refractory (r/r) RMS tumors. Subsequent administration of NK-92/5.28.z cells significantly enhanced antitumor activity in vitro. Applying recombinant TRAIL instead of NK-92/5.28.z cells confirmed that the synergistic antitumor effects of the combination treatment were mediated via TRAIL. Western blot analyses indicated that the combination treatment with bortezomib and NK-92/5.28.z cells increased apoptosis by interacting with the nuclear factor κB, JNK, and caspase pathways. Overall, bortezomib pretreatment can sensitize r/r RMS tumors to CAR- and, by upregulating DR5, TRAIL-mediated cytotoxicity of NK-92/5.28.z cells.

Keywords: MT: Regular Issue; NK cells; TRAIL; bortezomib; chimeric antigen receptor; immunotherapy; rhabdomyosarcoma.

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Conflict of interest statement

J.-H.K. has advisory roles for Bluebird Bio, Novartis, Roche, and Jazz Pharmaceuticals. T.T. and W.S.W. are named as inventors on patents and patent applications related to the therapeutic agent used in this study, owned by their respective academic institutions.

Figures

None
Graphical abstract
Figure 1
Figure 1
Determination of EC50 values of the aRMS cell lines RH30 and RH41 and the tumor organoid aRMS RMS335 in the presence of increasing bortezomib doses, shown using the luciferase toxicity assays (A and B) Luciferase-expressing RH30 cells were incubated for 24 h (A) and 48 h (B) with the indicated concentrations of bortezomib. After 24 and 48 h, luciferase was added to bortezomib-pretreated target cells and untreated controls. Cell lysis was calculated based on the luminescence signals of remaining cells and correlated with the signal of the untreated controls. (C and D) The EC50 values of RH41 (C) and RMS335 (D) were determined analogously after 24 h of treatment with the indicated bortezomib concentrations. The results of at least three independent experiments are shown. After 24 h incubation, the dose-response curve showed a corresponding effect on the RMS cells with EC50 values (dotted lines) in the nanomolar range.
Figure 2
Figure 2
Caspase-3 cleavage and caspase-3/-7 activity in aRMS target cells after bortezomib treatment aRMS cells were treated with different concentrations of bortezomib. (A) After 24 h, aRMS cells were lysed and caspase cleavage was assessed by western blot analysis: RH30 (left) and RH41(right). In addition to caspase cleavage, caspase activation was determined by Caspase-Glo assay. (B–D) For this purpose, RH30 (B), RH41 (C) and RMS335 (D) cells were treated with the indicated bortezomib concentrations for 24 h, and caspase-3/7 activity was quantified using a Caspase-Glo assay according to the manufacturer’s instructions. Caspase-3 was cleaved and activated in RH30 and RH41 cells but not in RMS335 cells. The results are presented as the mean ± standard deviation (SD), and one-way ANOVA with the Bonferroni method was used to evaluate differences. Differences for ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.001 were considered significant.
Figure 3
Figure 3
Bortezomib induces the expression of the TRAIL receptor DR5 but not DR4 aRMS cells were incubated for 24 h with the indicated concentrations of bortezomib. The cells were then stained with fluorescence-labeled antibodies against DR4 and DR5. DR4 and DR5 surface expression of the aRMS cell lines RH30, RH41, and RMS335 was analyzed by flow cytometry. Shown is a representative measurement from three independent experiments. A 24 h pretreatment with bortezomib increased the surface expression of DR5, but not DR4, in r/r aRMS cells in a dose-dependent manner.
Figure 4
Figure 4
Lysis of bortezomib-pretreated aRMS cells by NK-92/5.28.z cells as assessed by luciferase toxicity assays aRMS cells RH30 (A), RH41 (B), and RMS335 (C) were treated with the indicated bortezomib concentrations for 24 h. An equal number of cells were then seeded in 96-well plates and co-incubated with NK-92/5.28.z cells for 3 h at different effector-to-target (E:T) cell ratios. Luciferin was added to each well to quantify the luciferase signal of the remaining cells. Based on the signal of the untreated aRMS cells, cell lysis was calculated for each condition. The results of at least three independent experiments are shown. The combination of NK-92/5.28.z immunotherapy with bortezomib significantly increased the lysis of r/r aRMS cells compared to treatment with NK-92/5.28.z cells alone. The results are presented as the mean ± standard deviation (SD), and one-way ANOVA with the Bonferroni method was used to evaluate differences. Differences for ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.001 were considered significant.
Figure 5
Figure 5
Bortezomib and NK-92/5.28.z cells affect noncanonical NF-κB and JNK signaling in RMS qPCR analysis of p100, p65, JNK, and BCL-xL mRNA levels in RH30 (A) and RH41 (B) cells upon incubation with or without bortezomib and/or NK-92/5.28.z cells compared to untreated controls. Data are shown of three independent experiments. The results are presented as the mean ± standard deviation (SD), and one-way ANOVA with the Bonferroni method was used to evaluate differences. Differences for ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.001 were considered significant. (C) Western blot analysis of p100, p65, JNK, and the phosphorylated forms of these proteins and BCL-xL, as well as total and cleaved caspase-3, of RH30 and RH41 cells left untreated or treated with bortezomib and/or NK-92/5.28.z cells. GAPDH was used as loading control. Representative blots of two independent experiments are shown.
Figure 6
Figure 6
Synergistic interactions between bortezomib and TRAIL treatment Luciferase-based cytotoxicity assays were performed with bortezomib-pretreated aRMS cells and purified TRAIL protein. The results were used in a zero interaction potency (ZIP) model to analyze additive or synergistic effects. For RH30 cells (A), RH41 cells (B), and RMS335 cells (C), the ZIP reference for additive effects (top row) was modeled, showing the expected effects when bortezomib and TRAIL have an additive effect. In addition, the synergy score (bottom row) was used, indicating the actual interactions between the two drugs. Overall, the ZIP additive model calculated the expected response of a combination treatment with bortezomib and TRIAL based on the potency of the individual therapies and the sum of the individual effects (additive) without any interaction. The ZIP synergy score was calculated by comparing the observed combined effect of bortezomib and TRIAL with the expected individual effects (from the ZIP reference model). The observed combined effect of bortezomib and TRIAL was increased compared to the sum of the monotherapies, resulting in a positive score indicating a synergetic interaction between bortezomib and TRIAL.
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